With respect to peptides and proteins collected from nature, the amino acid sequences identified thereof are essential information in course of studying the biological properties and functions of the peptides and proteins. Currently, the full-length amino acid sequences for peptides and proteins are determined as deduced amino acid sequences, based on corresponding gene information thereof, that is, nucleotide sequences of c-DNAs produced from genomic genes or m-RNAs which encode their peptides. However, in identifying the c-DNAs produced from the genomic gene or m-RNA which encodes the peptide, the knowledge of partial amino acid sequences of the peptides is still required.
It is generally considered that, as the knowledge of the partial amino acid sequences of peptide, the N-terminal amino acid sequence and C-terminal amino acid sequence of peptide are particularly useful. Specifically explaining, for example, in selecting a c-DNA which encodes an intended peptide from a c-DNA library prepared from a large number of m-RNAs, if the N-terminal amino acid sequence and C-terminal amino acid sequence thereof are known, the aimed c-DNA can be selected by using nucleic acid probes that are produced based on the above amino acid sequences of the two terminals. Or, the aimed c-DNA can be amplified selectively by applying PCR with use of oligonucleotide primers that are produced based on the amino acid sequences of the two termini.
As the method for analyzing the N-terminal amino acid sequence of a peptide, there has been conventionally used a method of allowing an acid to act on a peptide to release the N-terminal amino acids successively by hydrolysis and identifying the amino acids resulted therefrom. Meanwhile, as the method for analyzing the C-terminal amino acid sequence of a peptide, there has been proposed a method of releasing the C-terminal amino acids thereof successively by chemical means and identifying the C-terminal amino acids released thereby, based on the molecular weight differences between the original peptide and truncated peptides that are obtained as reaction products therefrom. As the technique for releasing the C-terminal amino acids successively by chemical means, there is proposed, for example, a method comprising steps of allowing a vapor generated from a high concentration aqueous solution of pentafluoropropanoic acid (CF3CF2COOH) or a high concentration aqueous solution of heptafluorobutanoic acid (CF3CF2CF2COOH), to act on a dried peptide under heating up condition at 90° C., and releasing the C-terminal amino acids by selective hydrolysis enhanced with use of said perfluoroalkanoic acid [Tsugita, A. et al., Eur. J. Biochem. 206, 691-696 (1992)]. There is also proposed a method comprising steps of using, in place of the above high concentration aqueous solution of a perfluoroalkanoic acid, an acetonitrile solution of pentafluoropropanoic acid anhydride [(CF3CF2CO)2O] or an acetonitrile solution of heptafluorobutanoic acid anhydride [(CF3CF2CF2CO)2O], allowing a vapor generated form the solution, to act on a dried peptide under cooling down condition at such a low temperature, for example, at −18° C., and releasing the C-terminal amino acids selectively, which is forced with use of the perfluoroalkanoic acid anhydride [Tsugita, A. et al., Chem. Lett. 1992, 235-238; Takamoto K. et al., Eur. J. Biochem. 228, 362-372 (1995)].
In said technique for selectively releasing the C-terminal amino acids by allowing a perfluoroalkanoic acid or a perfluoroalkanoic acid anhydride, which are supplied as a vapor thereof, to act on a dried peptide, it has been reported that an oxazolone ring structure is once formed from the C-terminal amino acids as a reaction intermediate, in a dehydration reaction shown by the following reaction scheme (I):
then, the perfluoroalkanoic acid acts on the oxazolone ring to give rise to a reaction shown by the following reaction scheme (II):
as a result, reaction of selectively releasing the C-terminal amino acid therefrom is achieved.
As the above reaction of selectively releasing the C-terminal amino acid proceeds successively, there is obtained, at a timing when a given treatment time has passed, a mixture comprising a series of reaction products in which one to ten odd amino acid residues have been removed from the C-terminal of the original peptide, respectively. This mixture comprising a series of reaction products is subjected to mass spectrometry to measure the masses of the ion species derived from the reaction products, whereby can be obtained a series of peaks exhibiting the mass differences, which reflect the C-terminal amino acid sequence. Specifically explaining, the individual reaction products are formed in reaction of successively releasing C-terminal amino acids from the original peptide; hence, for example, a set of reaction products including several members in series, where up to several amino acid residues have been removed from the original peptide, are subjected to mass spectrometry and, thereby, the masses of corresponding ion species thereto can be analyzed collectively, which enables determination of C-terminal amino acid sequence of such several amino acid residues at one time.
Incidentally, for example, the information of C-terminal amino acid sequence used in production of nucleic acid probe or primer may ordinarily be, in terms of the nucleotide sequence which codes such amino acid sequence, about 18 to 24 bases and accordingly about 6 to 8 amino acids. The identification of C-terminal amino acid sequence of up to ten odd amino acid residues is required only in very rare cases. Therefore, the above methods for preparation of treated sample comprising a series of reaction products, in which all the removals extending up to 10 amino acid residues are included, by the reaction of releasing the C-terminal amino acids from the dried peptide, where a vapor of a perfluoroalkanoic acid or a perfluoroalkanoic acid anhydride are supplied in vapor phase and allowed to act thereon, are suitable for the above-mentioned purposes.